In general, the invention features methods and compounds for treating or preventing a cell death disease or inflammation.
A feature of many diseases, for example, neurodegenerative diseases, is the occurrence of cell death. While the death of any type of cell in the body as a result of a disease is a medical concern, the effects of the death of certain types of cells, for example neuronal cells, are particularly disturbing, as these cell types do not readily regenerate. Therefore, understanding the mechanisms that regulate cell death is essential to being able to prevent or treat conditions, such as neurodegenerative diseases.
Research has shown that caspases play a central role in the induction of apoptosis. For example, caspase-11 has been shown to modulate both cytokine production and apoptosis, and to be induced upon ischemic brain injury (Wang et al., J. Biol. Chem. 271:20580-20587, 1996; Wang et al., Cell 92:501-509, 1998). These studies suggest that caspase-11 is involved in mediating cell death and inflammatory responses. Discovery of a compound that modulates the expression of caspases and/or the occurrence of inflammation or cell death would provide a useful therapeutic for treating conditions involving inflammation or conditions in which cell death occurs, and also for preventing such conditions.
The present invention features methods and compounds for treating or preventing inflammation or a cell death disease. The methods involve the use of the plant extract wedelolactone and may be particularly useful for treating neurodegenerative diseases.
Accordingly, in a first aspect, the invention features a method for treating or preventing a cell death disease in a subject, comprising administering wedelolactone, or a derivative or salt thereof, to the subject. The cell death disease of the first aspect of the invention is not caused by hepatotoxicity.
In a second aspect, the invention features a method for treating or preventing inflammation in a subject, comprising administering wedelolactone, or a derivative or salt thereof, to the subject.
In one embodiment of the above aspects of the invention, the wedelolactone is present in an extract from a plant. Preferably the wedelolactone is substantially pure.
In a third aspect, the invention features a method for treating or preventing a cell death disease in a subject, involving administering to the subject a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
where R1 is selected from the group consisting of H, OH, and OCH3; and R2 is selected from the group consisting of H, CH3, and (CH2)xCH3, where x is a positive integer. This method is not used to treat a cell death disease caused by hepatotoxicity.
In a fourth aspect, the invention features a method for treating or preventing inflammation in a subject, involving administering to the subject a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
where R1 is selected from the group consisting of H, OH, and OCH3; and R2 is selected from the group consisting of H, CH3, and (CH2)xCH3, where x is a positive integer.
In a preferred embodiment of the third or fourth aspects of the invention, in the compound, R1 is OH and R2 is CH3.
In one embodiment of the first or third aspect of the invention, the cell death disease is a neurodegenerative disease, for example, ischemic brain injury or stroke. In another embodiment of any of the above aspects of the invention, the subject is a mammal, such as a human or a mouse.
In a fifth aspect, the invention features a chemical compound in a pharmaceutically acceptable carrier, having the formula: 
where R1 is selected from the group consisting of H, OH, and OCH3; and R2 is selected from the group consisting of H, CH3, and (CH2)xCH3, where x is a positive integer.
In a preferred embodiment of the fifth aspect of the invention, in the compound, R1 is OH and R2 is CH3.
In a sixth aspect, the invention features a method of synthesizing a 2,3-disubstituted benzo[b]furan, involving subjecting a molecule having the formula: 
to carbonylative heteroannulation.
In one embodiment of the sixth aspect of the invention, the molecule is in reaction with CO and CH3OH. In another embodiment, the carbonylative heteroannulation occurs in the presence of PdI2-thiourea, CBr4, and CsCO3.
In a seventh aspect, the invention features a method of synthesizing wedelolactone, involving subjecting a molecule having the formula: 
to lactonization.
By xe2x80x9ctreatingxe2x80x9d is meant to submit or subject an animal, cell, lysate or extract derived from a cell, or a molecule derived from a cell to a compound that decreases cell death or inflammation.
By a xe2x80x9ccell death diseasexe2x80x9d is meant a disease that results in the death of a cell or a population of cells. As used herein, the cell death disease is not caused by hepatotoxicity. The occurrence of cell death can be measured by determining cellular ATP levels, wherein a cell that is undergoing cell death has a decreased level of cellular ATP compared to a control cell. The occurrence of cell death may also be measured by staining with a vital dye, for example, trypan blue, where a cell that is dead will be stained with the vital dye, and a cell that is not dead will not be stained with the dye. The occurrence of cell death can also be measured by contacting a cell with Hoescht stain and viewing it for morphological indications of cell death. Such indications include nuclear fragmentation.
By a xe2x80x9cneurodegenerative diseasexe2x80x9d is meant a disease characterized by neuronal cell death. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer""s disease, Huntington""s disease and related polyglutamine expansion diseases, ischemic brain injury, stroke, amyotropic lateral sclerosis, multiple sclerosis, Lewy body disease, Menkes disease, Wilson disease, Creutzfeldt-Jakob disease, and Fahr disease.
By a xe2x80x9cneuronxe2x80x9d is meant a cell of ectodermal embryonic origin derived from any part of the nervous system of an animal. Neurons express well-characterized neuron-specific markers, including neurofilament proteins, MAP2, and class III tubulin. Included as neurons are, for example, hippocampal, cortical, midbrain dopaminergic, motor, sensory, sympathetic, septal cholinergic, and cerebellar neurons.
By a xe2x80x9cderivativexe2x80x9d is meant a structural derivative having a chemical modification of the compound that does not modify the ultimate level of cell death or inflammation, but that does enhance bioavailability, solubility, or stability in vivo or ex vivo or that reduces the toxicity or dosage required. Such modifications are known to those skilled in the field of medicinal chemistry. As used herein, a derivative of wedelolactone may be synthesized from the purified wedelolactone extract, or may be chemically synthesized using reagents other than the purified wedelolactone extract.
By xe2x80x9csubstantially purexe2x80x9d is meant a compound that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, chemical compound, e.g., wedelolactone. A purified compound may be obtained, for example, by high pressure liquid chromatograph, thin layer chromatography, or by synthesizing it.
By xe2x80x9cpreventing a cell death diseasexe2x80x9d is meant decreasing the number of cells that undergo cell death relative to an untreated control. As used herein, cell death may be inhibited. Preferably cell death is decreased 10% relative to a control compound with no activity in preventing cell death. More preferably cell death is decreased 50% relative to a control. Most preferably cell death is decreased 90% relative to a control.
By a xe2x80x9cplant extractxe2x80x9d is meant a compound or mixture of compounds that are obtained from a plant. A plant extract may be obtained from a plant, for example, by chopping up the plant into small pieces, treating the plant with high pressure, distilling the plant, or treating the plant with solvents.
By xe2x80x9ctreatingxe2x80x9d is meant submitting or subjecting an animal to a compound that promotes the elimination or reduction of a disease or condition or symptoms of a disease or condition, or that slows the progression of the disease. For example, an animal may be treated with plant extracts, synthesized organic molecules, naturally-occurring organic molecules, peptides, polypeptides, nucleic acid molecules, or components thereof.
By a xe2x80x9ccandidate modulatory compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate caspase-11 biological activity, or the occurrence of cell death or inflammation by employing one of the assay methods described herein. Compounds may include, for example, naturally-occurring organic molecules, synthesized organic molecules, polypeptides, nucleic acid molecules, or components thereof.
By xe2x80x9ccaspase-11 biological activityxe2x80x9d is meant any activity of caspase-11 found in cells. This includes the level of caspase-11 nucleic acids or polypeptides, and the effect of caspase-11 on substrates. This also includes the effect of caspase-11 on cell proliferation and cell death.
The present invention provides a number of advantages. For example, the methods described herein allow for the treatment or prevention of a cell death disease or inflammation. Substantially pure wedelolactone or a derivative or salt thereof, may be used to treat such conditions. A plant extract containing wedelolactone may also be used to treat or prevent inflammation or a cell death disease.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.